Changes in acetyl-CoA mediate Sik3-induced maturation of chondrocytes in endochondral bone formation.

The maturation of chondrocytes is strictly regulated for proper endochondral bone formation. Although recent studies have revealed that intracellular metabolic processes regulate the proliferation and differentiation of cells, little is known about how changes in metabolite levels regulate chondrocyte maturation. To identify the metabolites which regulate chondrocyte maturation, we performed a metabolome analysis on chondrocytes of Sik3 knockout mice, in which chondrocyte maturation is delayed. Among the metabolites, acetyl-CoA was decreased in this model. Immunohistochemical analysis of the Sik3 knockout chondrocytes indicated that the expression levels of phospho-pyruvate dehydrogenase (phospho-Pdh), an inactivated form of Pdh, which is an enzyme that converts pyruvate to acetyl-CoA, and of Pdh kinase 4 (Pdk4), which phosphorylates Pdh, were increased. Inhibition of Pdh by treatment with CPI613 delayed chondrocyte maturation in metatarsal primordial cartilage in organ culture. These results collectively suggest that decreasing the acetyl-CoA level is a cause and not result of the delayed chondrocyte maturation. Sik3 appears to increase the acetyl-CoA level by decreasing the expression level of Pdk4. Blocking ATP synthesis in the TCA cycle by treatment with rotenone also delayed chondrocyte maturation in metatarsal primordial cartilage in organ culture, suggesting the possibility that depriving acetyl-CoA as a substrate for the TCA cycle is responsible for the delayed maturation. Our finding of acetyl-CoA as a regulator of chondrocyte maturation could contribute to understanding the regulatory mechanisms controlling endochondral bone formation by metabolites.

Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum.

Ca2+ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)-biotin interaction and a copper-free click reaction system, we linked N3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors.

Engraftment of allogeneic iPS cell-derived cartilage organoid in a primate model of articular cartilage defect.

Induced pluripotent stem cells (iPSCs) are a promising resource for allogeneic cartilage transplantation to treat articular cartilage defects that do not heal spontaneously and often progress to debilitating conditions, such as osteoarthritis. However, to the best of our knowledge, allogeneic cartilage transplantation into primate models has never been assessed. Here, we show that allogeneic iPSC-derived cartilage organoids survive and integrate as well as are remodeled as articular cartilage in a primate model of chondral defects in the knee joints. Histological analysis revealed that allogeneic iPSC-derived cartilage organoids in chondral defects elicited no immune reaction and directly contributed to tissue repair for at least four months. iPSC-derived cartilage organoids integrated with the host native articular cartilage and prevented degeneration of the surrounding cartilage. Single-cell RNA-sequence analysis indicated that iPSC-derived cartilage organoids differentiated after transplantation, acquiring expression of PRG4 crucial for joint lubrication. Pathway analysis suggested the involvement of SIK3 inactivation. Our study outcomes suggest that allogeneic transplantation of iPSC-derived cartilage organoids may be clinically applicable for the treatment of patients with chondral defects of the articular cartilage; however further assessment of functional recovery long term after load bearing injuries is required.

Quality assessment tests for tumorigenicity of human iPS cell-derived cartilage.

Articular cartilage damage does not heal spontaneously and causes joint dysfunction. The implantation of induced pluripotent stem cell (iPSC)-derived cartilage (iPS-Cart) is one candidate treatment to regenerate the damaged cartilage. However, concerns of tumorigenicity are associated with iPS-Cart, because the iPSC reprogramming process and long culture time for cartilage induction could increase the chance of malignancy. We evaluated the tumorigenic risks of iPS-Cart using HeLa cells as the reference. Spike tests revealed that contamination with 100 HeLa cells in 150 mg of iPS-Cart accelerated the cell growth rate. On the other hand, 150 mg of iPS-Cart without HeLa cells reached growth arrest and senescence after culture, suggesting less than 100 tumorigenic cells, assuming they behave like HeLa cells, contaminated iPS-Cart. The implantation of 10,000 or fewer HeLa cells into joint surface defects in the knee joint of nude rat did not cause tumor formation. These in vitro and in vivo studies collectively suggest that the implantation of 15 g or less iPS-Cart in the knee joint does not risk tumor formation if assuming that the tumorigenic cells in iPS-Cart are equivalent to HeLa cells and that nude rat knee joints are comparable to human knee joints in terms of tumorigenicity. However, considering the limited immunodeficiency of nude rats, the clinical amount of iPS-Cart for implantation needs to be determined cautiously.

PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets.

We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.

A ratiometric fluorescent molecular probe for visualization of mitochondrial temperature in living cells.

Mitochondrial thermodynamics is the key to understand cellular activities related to homeostasis and energy balance. Here, we report the first ratiometric fluorescent molecular probe (Mito-RTP) that is selectively localized in the mitochondria and visualize the temperature. We confirmed that Mito-RTP could work as a ratiometric thermometer in a cuvette and living cells.

Techniques used in high-scoring and low-scoring 'Roche' vaults performed by elite male gymnasts.

The 16 highest-scored Roche vaults (G1) performed during the 2000 Olympic Games were compared with those receiving the 16 lowest-scores (G2). A 16-mm motion picture camera operating at 100 Hz recorded the vaults during the competition. The results of t tests (p < .05) indicated G1, compared to G2, had (a) shorter time of board support, greater normalised average upward vertical force and backward horizontal force exerted by the board, greater change in the vertical velocity while on the board, and greater vertical velocity at board take-off, (b) comparable linear and angular motions in pre-flight, (c) smaller backward horizontal impulse exerted by the horse, smaller loss of the horizontal velocity while on the horse, and greater horizontal and vertical velocities at horse take-off, (d) greater height and larger horizontal distance of post-flight, (e) higher body mass centre at knee release, and (f) higher mass centre, greater normalised moment of inertia, and smaller vertical velocity at mat touchdown. Therefore, gymnasts and coaches should focus on sprinting the approach; blocking and pushing-off the take-off board rapidly and vigorously; departing the board with a large vertical velocity; exerting large downward vertical force and small forward horizontal force from the hand-stand position while on the horse; departing the horse with large horizontal and vertical velocities; and completing the majority of the double salto forward near the peak of trajectory and releasing the knees above the top of the horse to prepare for a controlled landing.

A nanoparticle-based ratiometric and self-calibrated fluorescent thermometer for single living cells.

The homeostasis of body temperature and energy balance is one of the major principles in biology. Nanoscale thermometry of aqueous solutions is a challenging but crucial technique to understand the molecular basis of this essential process. Here, we developed a ratiometric nanothermometer (RNT) for intracellular temperature measurement in real time. Both the thermosensitive fluorophore, ß-diketonate chelate europium(III) thenoyltrifluoroacetonate, and the thermoinsensitive fluorophore, rhodamine 101, which was used as a self-reference, are embedded in a polymeric particle that protects the fluorophores from intracellular conditions. The ratiometric measurement of single RNT spots is independent of the displacement of the RNT along the z-axis. The temperature is therefore determined at the location of each RNT under an optical microscope regardless of the dynamic movement of living cells. As a demonstration of the spot-by-spot intracellular thermometry, we successfully followed the temperature change in individual RNT spots in a single cell together with the Ca(2+) burst induced by the Ca(2+) ionophore ionomycin. The temperature increases differently among different spots, implying heterogeneous heat production in the cell. We then show that, in some spots, the temperature gradually decreases, while in others it remains high. The average temperature elevation within a cell is positively correlated to the increase in Ca(2+), suggesting that the activity and/or number of heat sources are dependent on the Ca(2+) concentration.

Intracellular click reaction with a fluorescent chemical Ca2+ indicator to prolong its cytosolic retention.

The powerful strategy of "intracellular click reaction" was used to retain a chemical Ca(2+) indicator in the cytosol. Specifically, a novel clickable Ca(2+) indicator "N3-fura-2 AM" was coupled with dibenzylcyclooctyl-modified biomacromolecules via copper-free click reaction in living cells and Ca(2+) oscillation was observed for an extended period of time.

Walking nanothermometers: spatiotemporal temperature measurement of transported acidic organelles in single living cells.

We fabricated fluorescent nanoparticles which monitor temperature changes without sensitivity to pH (4-10) and ionic strength (0-500 mM). The nanothermometers spontaneously enter living HeLa cells via endocytosis, enclosed in acidic organelles, i.e., endosome/lysosome, and then transported along microtubules in a temperature-dependent manner, working as "walking nanothermometers".

The roche vault performed by elite gymnasts: somersaulting technique, deterministic model, and judges' scores.

The purpose of the study was to determine the mechanical variables that are related to successful post-flight somersaulting performance of the Roche vault. The 23 Roche vaults performed during the 2000 Olympic Games were filmed by a 16-mm camera operating at 100 Hz. The 2-D direct linear transformation technique was used for spatial calibration. Approximately 60 frames were digitized per vault. The method of Hay and Reid (1988) was used to develop a deterministic model to identify the mechanical variables that govern linear and angular motions of the vault. Correlational analysis was used to establish the strength of the relationship between the mechanical variables identified and the judges' scores. Significant correlations indicated that the higher judges' scores were negatively related to five mechanical variables and positively related to seventeen variables in the model. The normalized horizontal displacement of body center of mass (CM) from the knee grasp to the peak of post-flight was the best single predictor of the judges' score and accounted for 50% of variation in the judges' score. Finally, the landing point deductions and the official horizontal distance of post-flight collectively accounted for 86% of the variance in the judges' scores.

Somersaulting techniques used in high-scoring and low-scoring Roche vaults performed by male Olympic gymnasts.

The aim of this study was to compare the somersaulting techniques used in the 16 highest-scoring and 16 lowest-scoring Roche vaults. Our hypothesis was that the gymnasts performing the highest-scoring Roche vaults would demonstrate a better technique than those performing the lowest-scoring Roche vaults while on the horse (pushing off the horse more effectively), somersaulting (executing most of the required somersaults higher in flight), and landing (showing a greater control). A 16-mm motion picture camera, operating at 100 Hz, recorded the vaults during the official competition. The two-dimensional direct linear transformation was used for spatial reconstruction. The results of t-tests (P < 0.05) indicated that, compared with the low-scoring gymnasts, the high-scoring gymnasts had: (1) greater height of body centre of mass and a more fully extended body position at the horse take-off; (2) greater height of body centre of mass at the peak of post-flight, knee release, and touchdown on the mat; (3) greater horizontal and vertical displacements of body centre of mass, greater somersaulting rotation, and longer time from the knee release to mat touchdown; and (d) markedly smaller landing point deductions. In conclusion, a successful Roche vault is likely when the focus is on: (a) leaving the horse with a large vertical velocity in an extended body position to achieve a high trajectory of centre of mass by first extending the legs, then immediately pushing off the horse vigorously, using the muscles of the upper extremity; (b) grasping the knees immediately after the take-off from the horse, achieving the tightly tucked body position early during the ascent to the peak, and completing two-thirds of the required somersaults at a great height; (c) releasing the knees and extending the body above the top level of the horse; and (d) contacting the mat with a high body centre of mass position.

Phase-dependent chronotropic response of the heart during running in humans.

Heartbeat modulation by muscle contraction during rhythmic exercise involving a small muscle mass is phase-dependent, reflecting the timing of the muscle contraction within the cardiac cycle, but it remains unclear whether such modulation occurs during whole body exercise. To determine whether phase-dependent chronotropic changes in the heart would occur during running, we investigated the relationship between R-R interval (RRI) and the timing of vastus lateralis muscle contractions within the cardiac cycle. Seven healthy subjects were examined during high intensity running where the target heart rate was 160 beats . min(-1). The running pitch was made to wax and wane periodically in the neighborhood of the target heart rate to scan the effect of footfall timing within the cardiac cycle on heart period. We found that when muscle contraction occurred early in the cardiac cycle, RRI was reduced from the mean RRI (P<0.05). Conversely, when muscle contraction occurred in the latter half of the cardiac cycle, RRI tended to increase (P>0.05). Thus, the curve reflecting this phase-dependent relationship between heart period and timing of muscle contraction showed a positive slope within the first one-quarter to three-quarters of the cardiac cycle. Our results suggest the existence of a mechanism that provides beat-by-beat regulation of RRI even when it is very short (approximately 375 ms), i.e., a cardio-locomotor synchronization develops during running, when the frequencies of the two rhythms approach one another.

Comparison of cardio-locomotor synchronization during running and cycling.

By comparing the characteristics of cardiac-locomotor synchronization (CLS) in running and cycling individuals, we tested whether the characteristics of CLS occurring during rhythmic exercise adhere to the central origin hypothesis, which postulates a direct interaction between cardiovascular centers in the brain and the pattern generator in the spinal cord. Ten healthy subjects performed both exercises at the same intensity (150 beats.min(-1)) and cadence (150 steps.min(-1) during running and 75 rpm during cycling), while electrocardiograms and electromyograms from the right vastus lateralis muscle were monitored continuously. An examination of the occurrence of heart beats with respect to the locomotor phase revealed that, in running subjects, CLS exists for relatively prolonged periods at specific phases, whereas, in cycling subjects, it occurs intermittently and is not phase-specific [maximum duration of CLS: 113.6 (66.5) and 58.0 (29.3) s ( P<0.05), respectively]. Determining the probability of CLS by chance as a function of its duration, we also found that, during running, CLS likely results from entrainment, whereas, during cycling, it results from chance, occurring when the cardiac rhythm approached the locomotor rhythm. Our result indicated that the duration of muscle contraction during cycling [317.0 (18.1) ms] was significantly longer than during running [205.6 (20.2) ms]. These results indicated that the difference in the CLS characteristics between running and cycling might be influenced by differences in peripheral inputs between exercise modes.